This invention relates to the preparation and use of labeled oligonucleotides. More particularly, it relates to methods of preparing and using oligonucleotides wherein label-conjugated nucleotides are incorporated into a nucleic acid sequence in a defined repetitive manner which allows for optimum detectability.
Several publications are referenced in this application, with full citations appearing in the text of the specification. These references describe the state-of-the-art to which this invention pertains, and are hereby incorporated herein by reference.
Many different compounds have been used to detect nucleic acids. Broadly, labels of nucleic acids can be divided into two classes: (1) those which covalently modify nucleic acids with a detectable moiety, and (2) those which non-covalently modify nucleic acids with a detectable moiety, i.e., by ionic interactions, hydrogen-bonding, or intercalation. In general, non-covalent probes of nucleic acids exhibit dramatically increased detectability upon binding to nucleic acids, and consequently, have been very useful in assays designed to determine the total nucleic acid present in a given sample. In addition, non-covalently bound molecules can, and will, migrate from a labeled strand to an unlabeled one. Covalently bound molecules, on the other hand, can not migrate from a labeled oligonucleotide to an unlabeled one. Therefore covalently bound moieties are preferred for use as tagged nucleic acid probes.
Examples of compounds which have been covalently attached to nucleic acid sequences include conjugates between nucleotide triphosphates or phosphoramidites and labeling moieties, and directly reactive dyes, e.g. fluorescent moieties. Nucleotide triphosphates are incorporated into a nucleic acids by nucleic acid polymerases. Commercially available nucleotide triphosphates-dye conjugates include dCTP-Cy3, dCTP-Cy5, dUTP-FluorX, etc. available from DuPont, Molecular Probes, Boehringer Mannheim, and Amersham Life Sciences. Such dye conjugates contain cyanine or fluorescein derivatives which are covalently bound to the nucleotide, and each dye conjugate differs with respect to the absorbance maxima of the dye moiety. Directly reactive dyes covalently bind to an existing nucleic acid sequence. A few reactive dyes are commercially available, including various psoralens and ethidium mono- and di-azides.
The chemistry associated with conjugates of phosphoramidites and labeling moieties has dramatically improved in recent years allowing for the complete synthesis of labeled oligonucleotides with commercially available nucleic acid synthesizers. Labeled oligonucleotides have also been synthesized by a combination of modified phosphoramidites and reactive dyes, typically involving the incorporation of primary amines in the oligonucleotide during synthesis followed by covalent coupling of the amine groups to a reactive dye.
Of the three methods for the covalent linkage of labeling moieties to oligonucleotides, the nucleotide triphosphate-dye conjugates offer the greatest flexibility and the highest achievable specific detectability, i.e. fluorescence. Synthetic nucleic acids (molecules produced non-enzymatically) are generally limited to less than 100 bases and are subject to variable dye coupling chemistries. Directly reactive dyes, such as ethidium monoazide, react non-specifically and can potentially damage the labeled oligonucleotide. Polymerase-driven labeling, on the other hand, can produce molecules from a few tens of bases to several kilobases, can utilize standard labeling methods such as nick translation and primer extension reactions, and the degree of dye incorporation can be roughly controlled by varying the ratio of labeled NTP to unlabeled NTP.
The primary limitation of polymerase-driven labeling of nucleic acids is the absence of absolute control of the amount of label incorporated into a particular sequence. For example, if one desires to label DNA with dCTP-Cy3 and the specific sequence has only a limited number of xe2x80x9cCxe2x80x9d sites, then the resulting labeled oligonucleotide will have relatively few Cy3 molecules and consequently a low specific fluorescence. The present invention overcomes this sequence specific limitation and optimizes the incorporation of the label by polymerase.
The invention provides a method for the preparation and purification of optimally labeled oligonucleotides comprising the steps of (a) preparing a primer; (b) preparing a template oligonucleotide containing a nucleotide sequence complementary to the primer, and a nucleotide repeat region downstream from the complementary region; (c) annealing the template and primer in a suitable reaction medium containing a polymerase, nucleotide triphosphates and label-conjugated nucleotide triphosphates; (d) initiating synthesis of a complementary strand on the template; (e) attaching the oligonucleotide containing a target sequence adjacent to the complementary strand; and (f) purifying the optimally labeled oligonucleotide by any appropriate method.
The invention provides a method for preparing optimally labeled oligonucleotides comprising (a) preparing a template having a primer binding region, a 5xe2x80x2 extension region for the incorporation of label-conjugated nucleotide triphosphates, and a 3xe2x80x2 overhang region; and (b) labeling an oligonucleotide target sequence by denaturing the target sequence, adding excess template, dNTPs and polymerase in a suitable reaction medium.
The invention also provides an oligonucleotide comprising a nucleotide sequence complementary to a primer, and a nucleotide repeat region downstream from said complementary sequence, wherein the nucleotide repeat region comprises Nt where Nt is any nucleotide which can form a base pair with a label-conjugated nucleotide triphosphate, and optionally, a plurality of nucleotides, N, which are not capable of forming a base pair with a label-conjugated nucleotide triphosphate.
The invention further provides an optimally labeled oligonucleotide comprising a radiolabeled nucleic acid sequence and a nucleotide repeat region, wherein the nucleotide repeat region comprises Nf where Nf is any nucleotide which is conjugated to a label, and optimally, a plurality of nucleotides, N, which are not conjugated to a label.